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Biochemistry 1986, 25, 7 1 10-7 1 17
Membrane Channel Forming Polypeptides. Molecular Conformation and Mitochondrial Uncoupling Activity of Antiamoebin, an a-Aminoisobutyric Acid Containing Peptidet Manoj K. Das, S . Raghothama, and P. Balaram* Molecular Biophysics Unit and Sophisticated Instruments Facility, Indian Institute of Science, Bangalore 560 01 2, India Received March 20, 1986; Revised Manuscript Received July 14, 1986
I (Ac-Phe-Aib-Aib-Aib-DIva-Gly-Leu- Aib Aib-HypGln-D-Iva-HypAibPrePhol) have been investigated in dimethyl sulfoxide solution by one- and two-dimensional NMR techniques. A substantial number of resonances in the 270-MHz ‘H NMR spectrum have been assigned. Intramolecularly hydrogen-bonded (solvent inaccessible) NH groups have been identified by determining solvent and temperature dependence of NH chemical shifts and rates of hydrogen-deuterium exchange. Ten backbone NH groups are inaccessible to solvent, while three N H groups assigned to the Phe(l), Aib(2), and Aib(8) residues are exposed to solvent. Interresidue nuclear Overhauser effects are consistent with rc/ values of 120 f 30’ for Phe(1) and Leu(7). The N M R results, together with the stereochemical constraints imposed by the presence of a-aminoisobutyryl, isovalyl, prolyl, and 4-hydroxyprolyl residues, favor a highly ordered structure. Two backbone conformations consistent with the data are considered. Antiamoebin is shown to be an effective uncoupler of oxidative phosphorylation in rat liver mitochondria, providing evidence for its membrane-modifying activity. ABSTRACT: The conformations of the 16-residue fungal peptide antiamoebin
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Membrane-modifying peptides of fungal origin, which are rich in a-aminoisobutyric acid (Aib), have been the focus of several recent investigations (Mathew & Balaram, 1983a; Jung et al., 1981). Representative sequences are listed in Figure 1. Alamethicin, a 20-residue polypeptide, which forms voltage-gated channels across lipid bilayer membranes (Mueller & Rudin, 1967; Boheim & Kolb, 1978; Hall et al., 1984), is the most widely studied member of this class of peptides (Mathew & Balaram, 1983; Jung et al., 1981; Fox & Richards, 1982; Hall et al., 1984). The Aib-containing fungal peptides are characterized by a remarkable microheterogeneity of the natural products and occur as complex mixtures of closely related sequence analogues (Balasubramanian et al., 1981; Rinehart et al., 1981; Briickner et al., 1984; Briickner & Przybylski, 1984; Przybylski et al., 1984). Acetylation of the amino-terminal and the presence of a Cterminal @-aminoalcohol are common features of these sequences. The Aib-containing natural peptides may be broadly grouped into two classes: (i) the “long” sequences that contain 18-20 residues and lack hydroxyproline (Hyp), as exemplified by the alamethicins (Pandey et al., 1977a), suzukacillins (Katz et al., 1985), trichotoxins (Przybylski et al., 1984), hypelcins (Fujita et al., 1984), paracelsins (Przybylski et al., 1984), and trichorzianines (Bodo et al., 1985) and (ii) the “short” sequences that contain 15-16 residues and possess hydroxylated residues, particularly Hyp, as exemplified by the emerimicins (Pandey et al., 1977b), zervamicins (Rinehart et al., 1981), and antiamoebins (Pandey et al., 1 9 7 7 ~ ) . While alamethicin, suzukacillin, and trichotoxin have been shown to form stable conductance states in artificial lipid bilayers (Boheim et al., 1976; Boheim et al., 1978; Hall et al., 1984), definitive data have not been reported for the shorter peptides. However, references to the pore-forming activities of the antiamoebins, zervamicins, and emerimicins have appeared in the literature [see footnotes in Pandey et al. ‘Supported by a grant from the Department of Science and Technology, Government of India.
0006-2960/86/0425-7110$01.50/0
(1 977b,c) and Rinehart et al. (1 98 I)]. There are no reports on detailed structural studies on the shorter peptides. Conformational analysis of the short sequences assumes importance in view of the presence of as many as three Pro or Hyp residues in the C-terminal segments of zervamicins, emerimicins, and antiamoebins. These should interrupt intramolecular hydrogen bonding and may result in structures distinctly different from those inferred for the alamethicin C-terminal (Fox & Richards, 1982; Mathew & Balaram, 1983a; Bosch et al., 1985a; Banerjee et al., 1983). We describe in this paper an N M R study of antiamoebin I that permits the development of secondary structural models for this sequence. We also establish the membrane-modifying activity of this peptide by demonstrating its effectiveness as a mitochondrial uncoupler. MATERIALS AND METHODS Antiamoebin, a fungal peptide produced by the strains Emericellopsis poonensis Thirum., Emericellopsis synnematicola Mathur and Thirum., and Cephalosporium pimprina Thirum., was the kind gift of Dr. N. Narasimhachari, Medical College of Virginia, Virginia Commonwealth University, and was originally isolated at Hindustan Antibiotics, Pune, India, as described earlier (Thirumalachar, 1968). Reverse-phase high-performance liquid chromatography (HPLC) analysis of the peptide was carried out on a Lichrosorb RP-18 column (4 X 250 mm, 10-pm particle size) with gradient elution (65-85% MeOH-H20 in 20 min, 85-95% MeOH-H,O in 5 min, flow 0.8 mL m i d , detection 226 nm) on an LKB HPLC system. Purification of the peptide was effected by repetitive injections and collection of fractions using a Superrac fraction collector. For N M R studies, the peptide was used without HPLC purification (see Results and Discussion). ‘ H N M R spectra were recorded on a Bruker WH-270 FT-NMR spectrometer, equipped with an Aspect 2000 computer at the Sophisticated Instruments Facility, Indian Institute of Science, Bangalore. For correlated spectroscopy (COSY) (Figure 4), 512 free induction decays (FIDs), each of 24 accumulations, with 1 -s relaxation delay were collected. The 0 1986 American Chemical Society
MEMBRANE CHANNEL FORMING POLYPEPTIDES AiOmethiiin
I
VOL. 2 5 , NO. 22, 1986
7111
A c - A i b - P r o - A l b - A l o - A I b - A l o - G l n - A i b - V o l - A i b - G I ~ - L ~ U -Aib-Pra Vu1
Alb -Aib-Glu-Gln-Phol Suzukaclllln
Ac-Aib-Alo-Aib-Alo-AIb-Alo-Gln-Aib-Aib~Aib-Gly-t~u-Aib~P~o-Vol
Arb-Aib-Gln-Gln- Phol TrlchotoxinA-50
Ac-Aib-Gly-A~b-Leu-AIb-Gln-Alb-Aib-Aib-Alo-Alo-Aib-Pro-Leu-Aib-
D-lvo-Gln- Volol 28rvamicin I l A
Ac-Trp-lle-Gln-Aib-lle-Thr-Aib-Leu-Alb-
Hyp-Gln-Aib-Hyp-Alb-Pro-
Phol Antiomoebin I
.
Ac - P h e
- Aib-Aib-Aib-D-lvo-GIy-Le~-Alb-Aib-Uyp-Gln-D~lvo-HyD
Alb- Pro - Phol
FIGURE 1: Sequences of some Aib-containing membrane-modifying peptides. The sequences shown correspond to a single major component and are taken from the references mentioned in the text.
t l domain was zero filled to lK, while 1K data points were collected in the t2 domain resulting in a 1K X 1K data matrix. Phase-shifted sine bell window functions were applied prior to Fourier transformation on both domains. The spectral width in each dimension was 2500 Hz. Single channel detection with constant phase of pulses was used. In the difference nuclear Overhauser effect (NOE) experiments, the perturbed and normal spectra recorded sequentially (one on-resonance and one off-resonance) in different parts of the memory (8K of each) were obtained by low-power on-resonance saturation of a peak and by off-resonance shifting of the irradiation frequency, respectively. About 128 transients were accumulated with a relaxation delay of 3 s between transients to facilitate buildup of initial magnetization (Rao et al., 1983). Delineation of hydrogen-bonded N H groups was carried out as described earlier (Balaram, 1985; Kishore et al., 1985). Peptide effects on respiration of rat liver mitochondria were monitored with a Hansatech oxygen electrode as described earlier (Das et al., 1985).
RESULTSAND DISCUSSION Figure 2 shows an HPLC analysis of the sample of antiamoebin used in this study. A major component (-80%) is observed with a retention time of -21 min. A significant second component (18%) is seen at -20 min, while at least three minor components (1-2%) are also discernible. Four pure fractions with retention times of 20.4, 21.4, 23.7, and 24.7 min have been isolated by HPLC separation and shown to correspond to closely related polypeptides. The microheterogeneity of natural antiamoebin has been noted earlier (Pandey et al., 1977c; Rinehart et al., 1979). Sequence analogues of the antiamoebin I sequence, where the replacements of Pro for Hyp(l3), Aib for Iva(5), and Ala for Gly(6) occur, have been characterized by mass spectrometry (Pandey et al., 1978; Stroh et al., 1985). The 270-MHz ' H N M R spectrum of the purified major component (retention time 21.7 min) was practically indistinguishable from that of unpurified antiamoebin. All N M R studies were therefore carried out without further purification of the antiamoebin sample. The 270-MHz 'H N M R spectrum of antiamoebin in (CD3)*S0 is shown in Figure 3. The spectrum is fully consistent with the composition suggested for antiamoebin I (Figure 1, Pandey et al., 1 9 7 7 ~ ) .The amino acid analysis and 13CN M R spectrum of the peptide provide further confirmation (data not shown). Antiamoebin is largely insoluble in water, sparingly soluble in chloroform, and appreciably soluble in dimethyl sulfoxide. Conformational studies were therefore carried out in (CD3)2S0. Assignment of Resonances. Figure 4 shows the two-dimensional correlated spectrum (Aue et al., 1976; Wider et al., 1984) of antiamoebin in (CD3)2S0. The five expected connectivities between the N H and CaH resonances [Phe(l),
0
16
18 20 22 24 26 28
d Tirno(min) FIGURE 2: HPLC profile for antiamoebin (1 mg of peptide in 20 p L of MeOH). Conditions are as described under Materials and Methods.
Gly(6), Leu(7), Gln(1 l), Phol(l6)I are clearly seen. The Gly N H group is readily recognized by its triplet nature and its coupling to the resonance at 6 3.74 (C"H,), which in turn displays no further connectivity. The Gln spin system is characterized by the CsH2 (6 1.818)-CYH2 (6 2.187) connectivity, which permits identification of the Gln CaH (6 4.17) and N H (6 7.90) resonances. The Phe and Phol residues have overlapping CflH resonances in the region 6 2.55-3.00. The Phol C*H resonance is however observed at significantly higher field (6 3.818) as compared to the Phe C"H group (6 4.340). A similar chemical shift has been reported for Phol CaH in alamethicin (Martin & Williams, 1976). This resonance is also unambiguously identified by its connectivity to resonances at 6 3.2-3.5 (Phol CFH,), which in turn are coupled to a triplet OH resonance at 6 4.62. This hydroxyl proton resonance disappears on addition of D20. The entire Leu spin system cannot be unambiguously traced in the COSY spectrum due to overlap of the CBH, and CYH resonances (Nagayama & Wuthrich 1981; Davoust et al., 1983). However, the assignments of the C*H and N H protons are unequivocal, since all other residues with coupled C*H and N H resonances have been identified. The Hyp C"H and CYH resonances were assigned by virtue of their common connectivities to the C0H2 resonances, while the CYH protons were also coupled to the hydroxyl resonances at 6 5.18. The assignments are indicated in Figure 3, and the proton chemical shifts of unambiguously identified residues are summarized in Table I. An unambiguous assignment of the various singlet resonances to specific Aib/Iva residues is not possible at present. The singlet N H resonances at 6 6.78 and 7.29 correspond to the Gln carboxamide side-chain protons. A small geminal coupling between these protons is not detected in Figure 3, but is clearly observed as an off-diagonal cross peak in the COSY spectrum (Figure 4). In the low-field region, 15 N H resonances can be identified, due to 13 backbone and 2 side-chain N H groups. Resonances are labeled S,, D,, and T,, where S, D, and T denote singlets, doublets, and triplets, respectively, while the subscript n indicates the order of appearance from low field in (CD,),SO (Figure 5).
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6 IPPm) FIGURE 3: 270-MHz IH NMR spectrum of antiamoebin in dimethyl sulfoxide-d,, (CD,),SO (20 mg mL-I). Assignments indicated were derived from COSY experiments (see Figure 4).
Table I: Proton Chemical Shifts' for Specific Residues of
Antiamoebin in (CD,),SO
Table II: NMR Parameters" far Backbone N H Resonances in
Antiamoebin
chemical shifts, 6 residue
C"H others Phe(l) 4.33 2.95, 2.82 (COH) 7.29-7.22 (aromatic protons) 7.98 (T,) 3.64 Gly(6) Gln(ll) 7.86 (DJ 4.12 1.88 (CBH2) 7.14, 6.73 (-CONH) LO"(7) 7.67 (Ds) 4.03 1.65 (CIBH2,CrH) 0.90, 0.84 (C*HH,) Phol(l6) 7.20 (Dll) 3.73 3.00, 2.56 (COH) 3.43, 3.14 (C"H) 4.66 (-OH) 7.29-7.22 (aromatic protons) Hyp(l0, 13)' 4.51 5.13 (-OH), 4.28 ( O H ) 2.11. 1.77 (COH), 4.40 (C"H) 4.37 5.13 (-OH), 4.20 (CIH) Hyp(I0. 13)' 2.11, 1.67 (COH), 4.54 (C"H) 'AH Aib and Iva resonances a n n o t be assigned to specific residues in the sequence. The chemical shift values for these protons are therefore not tabulated. However, the A W ) NH and . W 8 ) NH resonances have been identified by NOE connectivity to the preceding C"H resonance (see text and Table I1 far 6 values). bTheassignment of the Hyp spin systems to specific residues in the sequence is sible at the present. NH 8.32 (D,)
Delineation of Hydrogen-Bonded NH Croups. The possible involvement of N H groups in intramolecular hydrogen bonding was probed with three criteria (Kessler, 1982; Balaram, 1985; Wiithrich, 1976): (i) temperature dependence of N H chemical shifts, (ii) solvent dependence of N H chemical shifts in CDC13-(CD3)2S0 mixtures, and (iii) hydrogendeuterium (H-D) exchange studies in (CD3)2SO-D20 mixtures. The temperature dependence of chemical shifts in (CD,),SO is linear for all the N H groups in antiamoebin (Figure 6). The chemical shifts (293 K) and temperature coefficients (d6/dT) are summarized in Table 11. Ten backbone resonances exhibit low d6/dT values (
